Methods and compositions for treatment of COPD diseases

ABSTRACT

The present disclosure relates to treatment of a pulmonary disease. The methods and kits provided herein facilitate relieving the symptoms resulting from the pulmonary disease (e.g., asthma, chronic obstructive pulmonary disease (COPD), etc.).

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/129,965, filed Sep. 28, 2016, entitled “Methodsand Compositions for Treatment of COPD Diseases”. U.S. application Ser.No. 15/129,965 is a 35 U.S.C. § 371 national phase application ofInternational Application Serial No. PCT/CN2015/076120, filed Apr. 9,2015 (WO 2015/158216 A1). International Application Serial No.PCT/CN2015/076120 claims priority to U.S. Provisional Patent ApplicationNo. 61/979,149, filed on Apr. 14, 2014. Each of the applicationreferenced above are hereby incorporated by reference in their entirety.

BACKGROUND

When a person inhales, air passes into lungs and flows throughprogressively smaller airways, which are surrounded by smooth muscles.When these smooth muscles are in a pathological condition (e.g.,swelling, constriction, etc.), various pulmonary diseases may resultsuch as, for example, asthma. Asthma affects about 300 million peopleworldwide, and current therapeutic methods for treating asthma includeimmunological modulation, anti-inflammation, and relaxation of airwaysmooth muscle. Hormones or their derivatives (e.g., glucocorticoid,beta-adrenogenic agonists) are generally used in these therapeuticmethods. However, uses of hormones and these derivatives might have riskof toxicity to liver, kidney and other organs.

SUMMARY

Embodiments of the present disclosure relate to a method for treatingdiseases, for example, a pulmonary disease. The method may includeadministering to a subject in need thereof a therapeutically effectiveamount of a compound of formula (I):

wherein the A, B, C, D, or E ring is independently fully saturated orpartially saturated, R¹ is selected from hydrogen or a carbohydrateresidue, C2, C11, C12, and C19 are each independently substituted withhydrogen or —OH, R^(2a) and R^(2b) are selected from hydrogen, —COOH, orCOOR⁴, or together form —CO2-, R⁴ is a certain monosaccharide residue,R^(3a) and R^(3b) together form CH₂, or are each independently selectedfrom —CH₃ or —CH₂—OH, as a stereoisomer, enantiomer or tautomer thereof,or a pharmaceutically acceptable salt thereof.

Embodiments of the present disclosure also relate to a kit for treatingdiseases, for example, a pulmonary disease. The kit may include anamount of the compound of formula (I), wherein the A, B, C, D, or E ringis independently fully saturated or partially saturated, R¹ is selectedfrom hydrogen or a carbohydrate residue, C2, C11, C12, and C19 are eachindependently substituted with hydrogen or —OH, R^(2a) and R^(2b) areselected from hydrogen, —COOH, or COOR⁴, or together form —CO2-, R⁴ is acertain monosaccharide residue, R^(3a) and R^(3b) together form CH₂, orare each independently selected from —CH₃ or —CH₂—OH, as a stereoisomer,enantiomer or tautomer thereof, or a pharmaceutically acceptable saltthereof. In certain embodiments, the compound of formula (I) is capableof treating the pulmonary disease. In certain embodiments, the compoundof formula (I) is in a pharmaceutically acceptable carrier.

In some embodiments, the disease may include a pulmonary disease, whichmay include at least one of asthma, chronic obstructive pulmonarydisease (COPD), bronchitis, chronic or acute bronchoconstriction, adultrespiratory distress syndrome, acute lung injury, and bronchiectasis. Inparticular embodiments, the pulmonary disease may include asthma orCOPD.

In some embodiments, the A and B rings are independently fullysaturated, and R^(2a) and R^(2b) together form —CO2-.

In some embodiments, the carbohydrate residue is a monosaccharideresidue or an oligosaccharide residue. In some embodiments, the certaincarbohydrate residue is a monosaccharide residue or an oligosaccharideresidue. In certain embodiments, the monosaccharide may be arabinose(Ara), glucuronic acid (GlcA) or 2-deoxy-glucuronic acid, glucose (Glc),or rhamnose (Rha). In certain embodiments, the oligosaccharide residuemay be a disaccharide residue, a trisaccharide residue, or atetrasaccharide residue.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C12 and C19 are eachindependently substituted with —OH, C15 and C16 are each independentlysubstituted with two hydrogens, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atrisaccharide residue. For example, the trisaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C12 and C19 are eachindependently substituted with —OH, C15 and C16 are each independentlysubstituted with two hydrogens, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, and R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atetrasaccharide residue. For example, the tetrasaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc-(1-2)-Glc.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C11 and C19 are eachindependently substituted with —OH, C11 is substituted with hydrogen,R^(2a) and R^(2b) together form —CO2-, R^(3a) and R^(3b) are —CH₃, andR¹ is a monosaccharide residue or an oligosaccharide residue. Inparticular embodiments, R¹ is a trisaccharide residue. For example, thetrisaccharide residue may be -Ara-[(1-2)-Rha]-(1-3)-Glc.

In certain embodiments, the A, B, and E rings are fully saturated, the Cand D rings are partially saturated, the C19 is substituted with —OH, C9is substituted with hydrogen, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, and R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atrisaccharide residue. For example, the trisaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc.

In some embodiments, the compound of formula (I) is isolated from anextract of kudingcha (KE). In some embodiments, the subject is a human.

In some embodiments, the method may include delivering thetherapeutically effective amount of the compound of formula (I) to anairway of a lung of the subject. In certain embodiments, the method mayinclude delivering the therapeutically effective amount of the compoundof formula (I) to an airway of a lung of the subject via inhalation ofthe compound of formula (I) by the subject.

In some embodiments, the kit may include a delivery device such as, forexample, a spray device or a pressurized delivery device.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D illustrates relaxation of airway smooth muscle by kudingchaextracts. After extraction with different solvents, the extracts weredissolved with equal volume of ethanol. The bronchial segments wereevoked by 10 μM MCh and relaxed with accumulated addition of extracts.FIG. 1A illustrates the extracts from n-butanol phase, FIG. 1Billustrates the extracts from ethyl acetate phase, FIG. 1C illustratesextracts from water phase, and FIG. 1D shows the quantitation for therelaxation (Bars represent mean±SEM, n=4).

FIGS. 2A-B illustrates that n-BuOH phase of kudingcha extracts releasesasthmatic airway resistance in mice. C57BL/6 adult mice were inducedwith OVA and challenged with methacholine. FIG. 2A illustrates thepercentage of airway resistance after inhaling n-BuOH phase andalbuterol, and FIG. 2B illustrates the quantitation of airway resistanceat 3 minutes after challenge (Bars represent mean±SEM, n=8).

FIGS. 3A-C illustrate partial purification for active ingredients ofkudingcha (Fr=the collected fraction after elution).

FIGS. 4A-F illustrate structures of partial identified compounds inkudingcha.

FIGS. 5A-D illustrate that Kudinoside A and D relaxe bronchial ringswith a dose-dependent manner. FIGS. 5A-B illustrate the reduced forceresponses of airway bronchial rings to Kudinoside D, while the airwaysmooth muscle is stimulated with methacholine. FIGS. 5C-D illustrate thereduced force responses of airway bronchial rings to Kudinoside A.

FIGS. 6A-B illustrate that Kudinoside D attenuates the airway resistancein acute asthma mouse model. FIG. 6A illustrates time course ofdecreased airway resistance within 5 min in response to PBS, 0.03 μgKudinoside D, 0.31 μg Kudinoside D, 3 μg Kudinoside D and 3 μg albuterolrespectively. FIG. 6B illustrates the statistical analysis of therelative percentage of airway resistance decreased at 3 min after givingthese reagents (**P<0.01 versus PBS. Bars represent mean±SEM).

FIG. 7 shows that Kudinoside D causes decrease in cytosolic calcium.Primary airway smooth muscle cells were stimulated with 100 μMacetylcholine (ACh) (upper panels), resulting elevation and maintenanceof calcium signals (arrow heads indicate typical alteration of thecells) for 8 minutes. After washing out ACh, new buffer containing 10 μMKudinoside D was added, and then stimulated with 100 μM acetylcholine. Asignificant reduction of calcium could be observed around 16 to 18minutes.

FIGS. 8A-C illustrate the relaxation of airway smooth muscle byKudinoside A, the Organic Phase and the Water Phase after HCl treatment.After HCl treatment, the reaction system was divided into Organic Phaseand Water Phase by extracted with CH₃Cl. Further, 5 μL of Organic Phaseand Water Phase, as well as 5 μL of 50 mM Kudinoside A dissolved in DMSOwere added in the chamber. The relaxation effect of these substances on10 μM MCh-evoked contraction was measured. FIG. 8A illustrates 50 μMKudinoside A, FIG. 8B illustrates the Organic Phase, and FIG. 8Cillustrates the Water Phase.

FIGS. 9A-B illustrate that L-type calcium channel may be a target ofKudinoside A. Whole-cell patch clamp recording of VDCC currents wereperformed under vehicle, or 50 μM Kudinoside A or 1 μM Nifedipinetreatment. FIG. 9A illustrates the recordings of three primary airwaysmooth muscle cells under vehicle, 50 μM Kudinosdie A and 1 μMNifedipine treatment, respectively. FIG. 9B illustrates thecurrent-voltage relationship for peak currents in Vehicle (circlecurve), 50 μM Compound A (squire curve) and 1 μM Nifedipine (trianglecurve).

DETAILED DESCRIPTION

Overview

Embodiments of the present disclose contemplate a use of kudingchaextracts and/or derivatives of the kudingcha extracts for treatingpulmonary diseases (e.g., asthma, chronic obstructive pulmonary disease(COPD), etc.) on a subject. The present disclosure relates, in part, tothe demonstration that kudingcha extracts and/or derivatives of thekudingcha extracts may relax smooth muscles of airways and relieve thesymptoms associated with pulmonary diseases on the subject. For example,as shown in the accompanying embodiments, kudingcha extracts (e.g.,kudinoside A, kudinoside D, etc.) may relax airway smooth muscle andreduce asthmatic constriction.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

As used herein, the term “alkyl” (alone or in combination with anotherterm(s)) refers to a saturated aliphatic hydrocarbon radical includingstraight chain and branched chain groups of 1 to 20 carbon atoms(whenever a numerical range; e.g. “1-20”, is stated herein, it meansthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbonatoms). Alkyl groups containing from 1 to 4 carbon atoms are referred toas lower alkyl groups. When said lower alkyl groups lack substituents,they are referred to as unsubstituted lower alkyl groups. Morepreferably, an alkyl group is a medium size alkyl having 1 to 10 carbonatoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl,tert-butyl, pentyl, and the like. Most preferably, it is a lower alkylhaving 1 to 4 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl,n-butyl, iso-butyl, or tert-butyl, and the like. The alkyl group may besubstituted or unsubstituted. When substituted, the substituent group(s)is preferably one or more, more preferably one to three, even morepreferably one or two substituent(s) independently selected from thegroup consisting of halo, hydroxy, unsubstituted lower alkoxy, aryloptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, aryloxyoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, 6-memberheteroaryl having from 1 to 3 nitrogen atoms in the ring, the carbons inthe ring being optionally substituted with one or more groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and the nitrogen atoms in the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 5- or 6-member heterocyclic grouphaving from 1 to 3 heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur, the carbon and nitrogen (if present) atomsin the group being optionally substituted with one or more groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups, mercapto, (unsubstituted lower alkyl)thio, arylthiooptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or alkoxy groups, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, RS(O)—, RS(O)₂—, —C(O)OR,RC(O)O—, and —NR₁₃R₁₄, wherein R₁₃ and R₁₄ are independently selectedfrom the group consisting of hydrogen, unsubstituted lower alkyl,trihalomethyl, cycloalkyl, heterocyclic and aryl optionally substitutedwith one or more, groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups.

Preferably, the alkyl group is substituted with one or two substituentsindependently selected from the group consisting of hydroxy, 5- or6-member heterocyclic group having from 1 to 3 heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur, the carbon andnitrogen (if present) atoms in the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 5-member heteroaryl having from 1 to3 heteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, the carbon and the nitrogen atoms in the group being optionallysubstituted with one or more groups, preferably one, two or three groupswhich are independently of each other halo, hydroxy, unsubstituted loweralkyl or unsubstituted lower alkoxy groups, 6-member heteroaryl havingfrom 1 to 3 nitrogen atoms in the ring, the carbons in the ring beingoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, or—NR₁₃R₁₄, wherein R₁₃ and R₁₄ are independently selected from the groupconsisting of hydrogen and alkyl. Even more preferably the alkyl groupis substituted with one or two substituents which are independently ofeach other hydroxy, dimethylamino, ethylamino, diethylamino,dipropylamino, pyrrolidino, piperidino, morpholino, piperazino, 4-loweralkylpiperazino, phenyl, imidazolyl, pyridinyl, pyridazinyl,pyrimidinyl, oxazolyl, triazinyl, and the like.

As used herein, the term “aromatic”, “ar” or “aryl” (alone or incombination with another term(s)) refer to aromatic cyclic groups (forexample 6 membered monocyclic, 10 membered bicyclic or 14 memberedtricyclic ring systems) which contain 6 to about 14 carbon atoms.Exemplary aromatic groups include phenyl, naphthyl, biphenyl, indenyl,and anthracene.

As used herein, the term “halogen” (alone or in combination with anotherterm(s)) refers to a fluorine substituent (“fluoro”, which may bedepicted as —F), chlorine substituent (“chloro”, which may be depictedas —Cl), bromine substituent (“bromo”, which may be depicted as —Br), oriodine substituent (“iodo”, which may be depicted as —I).

As used herein, the term “cycloalkyl” refers to a 3 to 8 memberall-carbon monocyclic ring, an all-carbon 5-member/6-member or6-member/6-member fused bicyclic ring or a multicyclic fused ring (a“fused” ring system means that each ring in the system shares anadjacent pair of carbon atoms with each other ring in the system) groupwherein one or more of the rings may contain one or more double bondsbut none of the rings has a completely conjugated pi-electron system.

Examples, without limitation, of cycloalkyl groups are cyclopropane,cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene,adamantane, cycloheptane, cycloheptatriene, and the like. A cycloalkylgroup may be substituted or unsubstituted. When substituted, thesubstituent group(s) is preferably one or more, more preferably one ortwo substituents, independently selected from the group consisting ofunsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, aryl optionally substituted with one or more, preferablyone or two groups independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, aryloxyoptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 6-member heteroaryl having from 1 to3 nitrogen atoms in the ring, the carbons in the ring being optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, unsubstituted lower alkyl or unsubstitutedlower alkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and nitrogen atoms of the group being optionally substituted withone or more, preferably one or two groups independently of each otherhalo, hydroxy, unsubstituted lower alkyl or unsubstituted lower alkoxygroups, 5- or 6-member heterocyclic group having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and nitrogen (if present) atoms in the group being optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, unsubstituted lower alkyl or unsubstitutedlower alkoxy groups, mercapto, (unsubstituted lower alkyl)thio, arylthiooptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, cyano, acyl, thioacyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro,N-sulfonamido, S-sulfonamido, RS(O)—, RS(O)₂—, —C(O)OR, RC(O)—, and—NR₁₃R₁₄ are as defined above.

As used herein, the term “alkenyl” (alone or in combination with anotherterm(s)) refers to a lower alkyl group, as defined herein, consisting ofat least two carbon atoms and at least one carbon-carbon double bond.Representative examples include, but are not limited to, ethenyl,1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

As used herein, the term “alkynyl” (alone or in combination with anotherterm(s)) refers to a lower alkyl group, as defined herein, consisting ofat least two carbon atoms and at least one carbon-carbon triple bond.Representative examples include, but are not limited to, ethynyl,1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

As used herein, the term “aryl” refers to an all-carbon monocyclic orfused-ring polycyclic (i.e., rings which share adjacent pairs of carbonatoms) groups of 1 to 12 carbon atoms having a completely conjugatedpi-electron system. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group may be substitutedor unsubstituted. When substituted, the substituted group(s) ispreferably one or more, more preferably one, two or three, even morepreferably one or two, independently selected from the group consistingof unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, mercapto, (unsubstituted lower alkyl)thio, cyano, acyl,thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, RS(O)—, RS(O)₂—,—C(O)OR, RC(O)—, and —NR₁₃R₁₄, with R₁₃ and R₁₄ as defined above.Preferably, the aryl group is optionally substituted with one or twosubstituents independently selected from halo, unsubstituted loweralkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedring (i.e., rings which share an adjacent pair of atoms) group of 5 to12 ring atoms containing one, two, or three ring heteroatoms selectedfrom N, O, or S, the remaining ring atoms being C, and, in addition,having a completely conjugated pi-electron system. Examples, withoutlimitation, of unsubstituted heteroaryl groups are pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline, purine and carbazole. The heteroaryl group maybe substituted or unsubstituted. When substituted, the substitutedgroup(s) is preferably one or more, more preferably one, two, or three,even more preferably one or two, independently selected from the groupconsisting of unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy,unsubstituted lower alkoxy, mercapto, (unsubstituted lower alkyl)thio,cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido,RS(O)—, RS(O)₂—, —C(O)OR, RC(O)—, and —NR₁₃R₁₄, with R₁₃ and R₁₄ asdefined above. Preferably, the heteroaryl group is optionallysubstituted with one or two substituents independently selected fromhalo, unsubstituted lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano,N-amido, mono or dialkylamino, carboxy, or N-sulfonamido.

As used herein, the term “Heterocyclic” refers to a monocyclic or fusedring group having in the ring(s) of 5 to 9 ring atoms in which one ortwo ring atoms are heteroatoms selected from N, O, or S(O)n (where n isan integer from 0 to 2), the remaining ring atoms being C. The rings mayalso have one or more double bonds. However, the rings do not have acompletely conjugated pi-electron system. Examples, without limitation,of unsubstituted heterocyclic groups are pyrrolidino, piperidino,piperazino, morpholino, thiomorpholino, homopiperazino, and the like.The heterocyclic ring may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two or three, even more preferably one or two,independently selected from the group consisting of unsubstituted loweralkyl, trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,mercapto, (unsubstituted lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, RS(O)—, RS(O)₂—, —C(O)OR,RC(O)—, and —NR₁₃R₁₄, with R₁₃ and R₁₄ as defined above. Preferably, theheterocyclic group is optionally substituted with one or twosubstituents independently selected from halo, unsubstituted loweralkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

Preferably, the heterocyclic group is optionally substituted with one ortwo substituents independently selected from halo, unsubstituted loweralkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

As used herein, the term “Hydroxy” refers to an —OH group.

As used herein, the term “Alkoxy” refers to both an —O-(unsubstitutedalkyl) and an —O-(unsubstituted cycloalkyl) group. Representativeexamples include, but are not limited to, e.g., methoxy, ethoxy,propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy,cyclohexyloxy, and the like.

As used herein, the term “Aryloxy” refers to both an —O-aryl and an—O-heteroaryl group, as defined herein. Representative examples include,but are not limited to, phenoxy, pyridinyloxy, furanyloxy, thienyloxy,pyrimidinyloxy, pyrazinyloxy, and the like, and derivatives thereof.

The terms “heterocycle”, “heterocyclic” or “heterocyclo” (alone or incombination with another term(s)) refer to fully saturated (i.e.,“heterocycloalkyl”), non-aromatic partially-saturated (i.e.,“heterocycloalkenyl”), or heterocylic aromatic (i.e. “heteroaryl”) ringstructure, typically having 3 to about 20 carbon atoms, more typicallyhaving 3 to about 14 carbon atoms. For example, the heterocyclic groupmay a 4 to about 7 membered monocyclic ring systems, a 7 to about 11membered bicyclic ring systems, or a 10 to about 15 membered tricyclicring systems, which have at least one heteroatom in at least one carbonatom-containing ring. Each ring of the heterocyclic group containing aheteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogenatoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfurheteroatoms may optionally be oxidized and the nitrogen heteroatoms mayoptionally be quaternized. The heterocyclic group may be attached at anyheteroatom or carbon atom of the ring or ring system.

A heterocyclyl may be a single ring, which typically contains from 3 to7 ring atoms, more typically from 3 to 6 ring atoms, and even moretypically 5 to 6 ring atoms. Examples of single-ring heterocyclylsinclude furanyl, thienyl (also known as “thiophenyl” and “thiofuranyl”),oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl(including 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl (also known as“azoximyl”), 1,2,5-oxadiazolyl (also known as “furazanyl”), and1,3,4-oxadiazolyl), pyrrolyl, pyrazolyl, imidazolyl, triazolyl,tetrazolyl, oxathiazolyl, oxatriazolyl (including 1,2,3,4-oxatriazolyland 1,2,3,5-oxatriazolyl), pyridinyl, diazinyl (including pyridazinyl(also known as “1,2-diazinyl”), pyrimidinyl (also known as“1,3-diazinyl”), and pyrazinyl (also known as “1,4-diazinyl”)),triazinyl (including s-triazinyl (also known as “1,3,5-triazinyl”),as-triazinyl (also known 1,2,4-triazinyl), and v-triazinyl (also knownas “1,2,3-triazinyl”)), oxathiazinyl (including 1,2,5-oxathiazinyl and1,2,6-oxathiazinyl), oxepinyl, thiepinyl, dihydrofuranyl,tetrahydrofuranyl, dihydrothienyl (also known as “dihydrothiophenyl”),tetrahydrothienyl (also known as “tetrahydrothiophenyl”), isopyrrolyl,pyrrolinyl, pyrrolidinyl, isoimidazolyl, imidazolinyl, imidazolidinyl,pyrazolinyl, pyrazolidinyl, dithiolyl, oxathiolyl, oxathiolanyl,oxazolidinyl, isoxazolidinyl, thiazolinyl, isothiazolinyl,thiazolidinyl, isothiazolidinyl, dioxazolyl (including 1,2,3-dioxazolyl,1,2,4-dioxazolyl, 1,3,2-dioxazolyl, and 1,3,4-dioxazolyl), pyranyl(including 1,2-pyranyl and 1,4-pyranyl), dihydropyranyl,tetrahydropyranyl, piperidinyl, piperazinyl, oxazinyl (including1,2,3-oxazinyl, 1,3,2-oxazinyl, 1,3,6-oxazinyl (also known as“pentoxazolyi”), 1,2,6-oxazinyl, and 1,4-oxazinyl), isoxazinyl(including o-isoxazinyl and p-isoxazinyl), oxadiazinyl (including1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, azepinyl, anddiazepinyl.

Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl,pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl,imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl,thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl,furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl,azepinyl, 4-piperidonyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane andtetrahydro-1,1-dioxothienyl, triazolyl, triazinyl, and the like.

A heterocyclyl alternatively may be from 2 to 5 (more typically from 2or 3) rings fused together, such as, for example, indolizinyl,pyranopyrrolyl, purinyl, imidazopyrazinyl, imidazolopyridazyl,pyridopyridinyl (including pyrido[3,4-b]-pyridinyl,pyrido[3,2-b]-pyridinyl, pyrido[4,3-b]-pyridinyl, and naphthyridinyl),pteridinyl, pyridazinotetrazinyl, pyrazinotetrazinyl,pyrimidinotetrazinyl, pyrindinyl, pyrazolopyrimidinyl,pyrazolopyrazinyl, pyrazolopyridazyl, or 4H-quinolizinyl. In someembodiments, the multi-ring heterocyclyls are indolizinyl,pyranopyrrolyl, purinyl, pyridopyridinyl, pyrindinyl, and4H-quinolizinyl.

Exemplary bicyclic heterocyclic groups include indolyl, benzothiazolyl,benzoxazolyl, benzodioxolyl, benzothienyl, quinuclidinyl, quinolinyl,tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl,indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl,cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (suchas furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyll or furo[2,3-b]pyridinyl),dihydroisoindolyl, dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl), tetrahydroquinolinyl and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.

Other examples of fused-ring heterocyclyls include benzo-fusedheterocyclyls, such as, for example, benzofuranyl (also known as“coumaronyl”), isobenzofuranyl, benzoxazolyl, benzoisoxazolyl (alsoknown as “indoxazinyl”), anthranilyl, benzothienyl (also known as“benzothiophenyl”, “thionaphthenyl” and “benzothiofuranyl”),isobenzothienyl (also known as “isobenzothiophenyl”, “isothionaphthenyl”and “isobenzothiofuranyl”), benzothiazolyl, benzoisothiazolyl,benzothiadiazolyl, benzoxadiazolyl, indolyl, isoindazolyl (also known as“benzopyrazolyl”), benzoimidazolyl, benzotriazolyl, benzazinyl(including quinolinyl (also known as “1-benzazinyl”) and isoquinolinyl(also known as “2-benzazinyl”)), phthalazinyl, quinoxalinyl,benzodiazinyl (including cinnolinyl (also known as “1,2-benzodiazinyl”)and quinazolinyl (also known as “1,3-benzodiazinyl”)),benzoimidazothiazolyl, carbazolyl, acridinyl, isoindolyl, indoleninyl(also known as “pseudoindolyl”), benzodioxolyl, chromanyl, isochromanyl,thiochromanyl, isothiochromanyl, chromenyl, isochromenyl, thiochromenyl,isothiochromenyl, benzodioxanyl, tetrahydroisoquinolinyl, benzoxazinyl(including 1,3,2-benzoxazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,and 3,1,4-benzoxazinyl), benzoisoxazinyl (including 1,2-benzisoxazinyland 1,4-benzisoxazinyl), benzoxadiazinyl, and xanthenyl. In someembodiments, the benzo-fused heterocyclyls are benzofuranyl,isobenzofuranyl, benzoxazolyl, benzoisoxazolyl, anthranilyl,benzothienyl, isobenzothienyl, benzothiazolyl, benzothiadiazolyl,benzoxadiazolyl, indolyl, isoindazolyl, benzoimidazolyl, benzotriazolyl,benzazinyl, phthalazinyl, quinoxalinyl, benzodiazinyl, carbazolyl,acridinyl, isoindolyl, indoleninyl, benzodioxolyl, chromanyl,isochromanyl, thiochromanyl, benzodioxanyl, tetrahydroisoquinolinyl,benzoxazinyl, benzoisoxazinyl, and xanthenyl.

As used herein, the term “heteroaryl” (alone or in combination withanother term(s)) refers to an aromatic heterocyclyl typically containingfrom 5 to 14 ring atoms. A heteroaryl may be a single ring or multiple(typically 2 or 3) fused rings. Such moieties include, for example,5-membered rings such as furanyl, thienyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, pyrazolyl,imidazolyl, triazolyl, tetrazolyl, oxathiazolyl, and oxatriazolyl;6-membered rings such as pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl, and oxathiazinyl; 7-membered rings such as oxepinyl andthiepinyl; 6/5-membered fused-ring systems such as benzofuranyl,isobenzofuranyl, benzoxazolyl, benzoisoxazolyl, anthranilyl,benzothienyl, isobenzothienyl, benzothiazolyl, benzoisothiazolyl,benzothiadiazolyl, indolizinyl, pyranopyrrolyl, benzoxadiazolyl,indolyl, isoindazolyl, benzoimidazolyl, benzotriazolyl, purinyl,imidazopyrazinyl, and imidazolopyridazyl; and 6/6-membered fused-ringsystems such as quinolinyl, isoquinolinyl, pyridopyridinyl,phthalazinyl, quinoxalinyl, benzodiazinyl, pteridinyl,pyridazinotetrazinyl, pyrazinotetrazinyl, pyrimidinotetrazinyl,benzoimidazothiazolyl, carbazolyl, and acridinyl. In some embodiments,the 5-membered rings include furanyl, thienyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, oxadiazolyl, pyrazolyl, and imidazolyl; the6-membered rings include pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,and triazinyl; the 6/5-membered fused-ring systems include benzoxazolyl,benzoisoxazolyl, anthranilyl, benzothienyl, isobenzothienyl, andpurinyl; and the 6/6-membered fused-ring systems include quinolinyl,isoquinolinyl, and benzodiazinyl.

Exemplary heteroaryl groups include pyrrolyl, pyrazolyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furyl,thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazolyl, triazinyl, and the like.

As used herein, the term “hydrogen” (alone or in combination withanother term(s)) refers to a hydrogen substituent and may be depicted as—H.

As used herein, the term “hydroxy” (alone or in combination with anotherterm(s)) refers —OH.

As used herein, the term “nitro” (alone or in combination with anotherterm(s)) refers to —NO₂.

As used here, “carbohydrate” is a compound containing one or moremonosaccharide residues. A monosaccharide may be a polyhydroxy aldehydesuch as D-glucose or a polyhydroxy ketone such as D-fructose.Monosaccharides may be classified according to the number of carbonsthey contain: monosaccharides with three carbons are trioses, those withfour carbons are tetroses, those with five carbons are pentoses, andthose with six and seven carbons are hexoses and heptoses, respectively.For example, a six-carbon polyhydroxy aldehyde such as D-glucose is analdohexose, whereas a six-carbon polyhydroxy ketone such as D-fructoseis a ketohexose. Monosaccharide may be present in differentdiasteromeric forms, such as α or β anomers, and D or L isomers. In someembodiments, monosaccharides may include unsubstituted saccharides suchas glucose or galactose, as well as modified saccharides in which one ormore hydroxyl groups contain substitutions or have been replaced withhydrogen or substituted carbon atoms (e.g., sialic acid)

An “oligosaccharide” refers to a short chain of covalently linkedmonosaccharide units (i.e., 2 to 9 monosaccharide units). For example,oligosaccharides include disaccharides each including two monosaccharideunits, trisaccharides each including three monosaccharide units, andtetrasaccharide each including four monosaccharide units. A“polysaccharide” consists of long chains of covalently linkedmonosaccharide units (i.e., more than 10 monosaccharide units). Aoligosaccharide and polysaccharide may include linear chain or branchedchains with various combinations of monosaccharide units. In particularembodiments, the linear chain or branched chains with variouscombinations of Glc, Ara, Rha and GlcA, wherein Glc is glucose, Ara isarabinose, GlcA is glucuronic acid, and Rha is rhamnose.

Portions of a carbohydrate molecule may include non-saccharide groupssuch as, for example, anti-idiotypic antibodies or cyclohexanederivatives that mimic the structure of a monosaccharide residue. Forexample, a compound that mimics a structure of monosaccharide residuesbut that contains few or no monosaccharide residues may be determinedusing a computer three dimensional modeling programs such as, forexample, the model of Hricouini et al., Biochem. 31:10018-10023 (1992).As used here, a monosaccharide residue may include a monosaccharideresidue or a non-saccharide group that mimics the structure of themonosaccharide residue. A disaccharide residue may include adisaccharide residue or a non-saccharide group that mimics the structureof the disaccharides residue. An oligosaccharide residue may include aoligosaccharide residue or a non-saccharide group that mimics thestructure of the oligosaccharides residue.

As used herein, the term “substitution” refers to a compound having asubstituent comprising at least one carbon, nitrogen, oxygen, or sulfuratom that is bonded to one or more hydrogen atoms. If a substituent isdescribed as being “substituted”, a non-hydrogen substituent is in theplace of a hydrogen on a carbon, nitrogen, oxygen, or sulfur of thesubstituent. Thus, for example, a substituted alkyl substituent is analkyl substituent wherein at least one non-hydrogen substituent is inthe place of a hydrogen on the alkyl substituent. To illustrate,monofluoroalkyl is alkyl substituted with a fluoro, and difluoroalkyl isalkyl substituted with two fluoros. It should be recognized that ifthere are more than one substitutions on a substituent, eachnon-hydrogen substituent may be identical or different (unless otherwisestated).

If a substituent is described as being “optionally substituted”, thesubstituent is either (1) substituted, or (2) not substituted. When themembers of a group of substituents are described generally as beingoptionally substituted, any atom capable of substitution in each memberof such group may be (1) substituted, or (2) not substituted. Such acharacterization contemplates that some members of the group are notsubstitutable. Atoms capable of substitution include, for example,carbon bonded to at least one hydrogen, oxygen bonded to at least onehydrogen, sulfur bonded to at least one hydrogen, or nitrogen bonded toat least one hydrogen. On the other hand, hydrogen alone, halogen, oxo,and cyano do not fall within the definition of being capable ofsubstitution.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals. In some embodiments, a pharmaceuticallyacceptable carrier may include one or more inactive pharmaceuticalingredients. The inactive pharmaceutical ingredients in thepharmaceutically acceptable carrier system may include stabilizers,preservatives, additives, adjuvants, aerosols, compressed air or othersuitable gases, or other suitable inactive pharmaceutical ingredientsformulated with the therapeutic compound (i.e., an active ingredient(API)).

The pharmaceutically acceptable carrier (e.g., inhalation carrier) mayinclude the pharmaceutically suitable inactive ingredients known in theart for use in various inhalation dosage forms, such as aerosolpropellants (e.g., hydrofluoroalkane propellants), surfactants,additives, suspension agents, solvents, stabilizers and the like.Alcohol is a good example of an ingredient that may be considered eitheractive or inactive depending on the product formulation.

As used herein, inhalation dosage forms include, but are not limited to,an aerosol being a drug product that is packaged under pressure andcontains the API and carrier system that are released upon activation ofan appropriate valve system intended for topical application to theolfactory epithelium. The inhalation dosage form may also be deliveredto mouth (lingual and sublingual aerosols), or lungs (inhalationaerosols); foam aerosol being a dosage form containing one or more APIs,surfactants, aqueous or nonaqueous liquids, and the propellants, wherebyif the propellant is in the internal (discontinuous) phase (i.e., of theoil-in-water type), a stable foam is discharged, and if the propellantis in the external (continuous) phase (i.e., of the water-in-oil type),a spray or a quick-breaking foam is discharged; metered aerosol being apressurized dosage form for use with metered dose valves which allow forthe delivery of a uniform quantity of spray upon each activation; powderaerosol being a product that is packaged under pressure and containsAPIs, in the form of a powder, that are released upon activation of anappropriate valve system; and, aerosol spray being an aerosol productwhich utilizes a compressed gas as the propellant to provide the forcenecessary to expel the product as a wet spray and being applicable tosolutions of medicinal agents in aqueous solvents.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but not limited to,hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, 5 ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of thedisclosure. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the disclosurewith one or more molecules of solvent. The solvent may be water, inwhich case the solvate may be a hydrate. Alternatively, the solvent maybe an organic solvent. Thus, the compounds of the present disclosure mayexist as a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the disclosure may be truesolvates, while in other cases, the compound of the disclosure maymerely retain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe disclosure and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Substantially” means nearly totally or completely, for instance, 95%,96%, 97%, 98%, 99% or greater of some given quantity.

As used herein, the terms “treating” and “treatment” are used to referto administration or application of a therapeutic agent to a subject orperformance of a procedure or modality on a subject for the purpose ofobtaining a therapeutic benefit of a disease or health-relatedcondition, and includes: (1) preventing the disease or condition fromoccurring in a mammal, in particular, when such mammal is predisposed tothe condition but has not yet been diagnosed as having it; (2)inhibiting the disease or condition, i.e., arresting its development;(3) relieving the disease or condition, i.e., causing regression of thedisease or condition; or (4) relieving the symptoms resulting from thedisease or condition, i.e., relieving pain without addressing theunderlying disease or condition. For example, treatment of a subjectwith asthma or COPD may include reducing a frequency, and/or severity ofone or more symptoms (e.g., dyspnea, wheezing, cough, chest discomfort,nasal congestion, etc.) associated with asthma, COPD, etc.

As used herein, the terms “preventing,” “inhibiting,” “reducing” or anyvariation of these terms, includes any measurable decrease or completeinhibition to achieve a desired result. For example, there may be adecrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range derivabletherein, reduction of activity or symptoms, compared to normal.

As used herein, the terms “disease” and “condition” may be usedinterchangeably or may be different in that the particular malady orcondition may not have a known causative agent (so that etiology has notyet been worked out) and it is therefore not yet recognized as a diseasebut only as an undesirable condition or syndrome, wherein a more or lessspecific set of symptoms have been identified by clinicians. Forexample, a disease and/or condition in the present disclosure mayinclude asthma, chronic or acute bronchoconstriction, chronicbronchitis, small airways obstruction, and emphysema, obstructive orinflammatory airways diseases (e.g., chronic eosinophilic pneumonia,chronic obstructive pulmonary disease (COPD), etc.), bronchitis, acutelung injury, bronchiectasis, etc.

As used herein, the terms “administered” and “delivered” are used todescribe the process by which a composition of the present disclosure isadministered or delivered to a subject, a target (e.g., a cell, atissue, an organ, a portion of a system, etc.) or are placed in directjuxtaposition with the target. In some embodiments, “administrated” and“delivered” may include a process that inhalation of a subject such thata composition of the present disclosure is delivered to the subject, atarget (e.g., a cell, a tissue, an organ, a portion of a system, etc.)or are placed in direct juxtaposition with the target. In someembodiments, a composition of the present disclosure may be administeredorally, intravenously, intraperitoneally, subcutaneously,intramuscularly, intrathecally, intradermally, nasally, enterically,pessaries, suppositories. For example, a target may be an airway smoothmuscle fiber or a portion of an airway of a subject. The terms“administered” and “delivered” are used interchangeably.

As used herein, the terms “contacted” and “exposed” when applied to atarget (e.g., a cell, a tissue, an organ, etc.), are used to describethe process by which a compound of the present disclosure isadministered or delivered to a target or are placed in directjuxtaposition with the target. The terms “administered” and “delivered”are used interchangeably with “contacted” and “exposed”.

As used herein, the terms “patient”, “subject” and “individual” are usedinterchangeably herein, and mean a mammalian subject to be treatedand/or to obtain a biological sample from the mammalian subject. Themammalian includes humans and domestic animals, such as cats, dogs,swine, cattle, sheep, goats, horses, rabbits, and the like.

As used herein, the term “effective” means adequate to accomplish adesired, expected, or intended result. For example, an “effectiveamount” may be an amount of a compound sufficient to produce atherapeutic benefit.

As used herein, the terms “therapeutically effective” or“therapeutically beneficial” refers to anything that promotes orenhances the well-being of the subject with respect to the medicaltreatment of a condition. This includes, but is not limited to, areduction in the onset, frequency, duration, or severity of the signs orsymptoms of a disease.

As used herein, the term “therapeutically effective amount” is meant anamount of a composition as described herein effective to yield thedesired therapeutic response. The amount of a compound of the disclosurewhich constitutes a “therapeutically effective amount” will varydepending on the compound, the condition and its severity, and the ageof the mammal to be treated, but can be determined routinely by one ofordinary skill in the art having regard to her own knowledge and to thisdisclosure.

As used herein, “methods known to one of ordinary skill in the art” maybe identified through various reference books and databases. Suitablereference books and treatise that detail the synthesis of reactantsuseful in the preparation of compounds of the present invention, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations”, 2ndEd., Academic Press, New York. 1983; H. O. House, “Modem SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist. “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; 1. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley Interscience, New York, 1992. Specificand analogous reactants may also be identified through the indices ofknown chemicals prepared by the Chemical Abstract Service of theAmerican Chemical Society’ which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., www.acs.org may be contacted formore details). Chemicals that are known but not commercially availablein catalogs may be prepared by custom chemical synthesis houses, wheremany of the standard chemical supply houses (e.g., those listed above)provide custom synthesis services.

As used herein, the terms “diagnostic”, “diagnose” and “diagnosed” meanidentifying the presence or nature of a pathologic condition.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used as described herein. Thespecific safe and effective amount or therapeutically effective amountwill vary with such factors as the particular condition being treated,the physical condition of the patient, the type of mammal or animalbeing treated, the duration of the treatment, the nature of concurrenttherapy (if any), and the specific formulations employed and thestructure of the compounds or its derivatives.

Multiple compounds of the disclosure have a central nucleus of fiverings (i.e., a core structure), designated herein as A, B, C, D, and Eas shown below:

The carbons of the central nucleus are numbered as set forth above. Forpurposes herein, the carbon at position 1 of the central nucleus isindicated herein as C 1, and so forth. In the compounds of thedisclosure, unless otherwise indicated, each of rings A, B, C, D and Eis independently fully saturated, partially saturated or fullyunsaturated. That is, hydrogens attached to any of the carbons atpositions 1-22 may be omitted so as to allow unsaturation within the A,B, C, D, and/or E rings.

The compounds of the disclosure, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present disclosure is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, such as HPLC using a chiralcolumn. When the compounds described herein contain olefinic doublebonds or other centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Zgeometric isomers. Likewise, all tautomeric forms are also intended tobe included.

The nomenclature used herein for the compounds of the disclosure is amodified form of the I.U.P.A.C. nomenclature system, using theChemDoodle Version 6.0.0 software program, wherein multiple compoundsare named herein as derivatives of the core structure described above.In addition, the configuration of the substituents are indicated in thenames of the compounds by an “α” if the substituent is below the planeof the indene ring and by a “β” is the substituent is above the plane ofthe indene ring. For example, a compound of formula (Ia) (showing thenumbering of the carbons):

Wherein the A, B, D, and E ring are independently fully saturated; the Cring is partially saturated; C2, C3, C5, C9, C10, C11, C12, C18, and C20are each independently substituted with hydrogen; C1, C7, C8, C11, C15,C16, C19, C21, and C22 are substituted with two hydrogens; R¹ and R^(2a)each are independently hydrogen; C2 is substituted with —OH, R^(2b) is—COOH, and R^(3a) and R^(3b) together form methylene, i.e., a compoundof the following formula (Ib):

is named herein as(1S,2R,5S,8S,9R,14R,15R,17R)-10,11-Dihydroxy-1,2,6a,6b,12a-pentamethyl-9-methylene-2,3,4,4a,5,6,6a,6b,7,8,8a,10,11,12,12a,12b,13,14b-octadecahydro-1H-picene-4a-carboxylicacid. The common name of the compound is Ilekudinol B with a ChemSpiderdatabase ID: 8822838.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present disclosure contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are non-superimposablemirror images of one another.

Tautomers refer to various forms of a compound that differ only by theshifting of one or more double bonds and the concomitant shifting ofhydrogen atoms. The present disclosure includes tautomers of any saidcompounds.

Methods and Compositions for Treatment of Diseases

Embodiments of the present disclosure relate to a method for treatingdiseases, for example, a pulmonary disease. The method may includeadministering to a subject in need thereof a therapeutically effectiveamount of a compound of formula (I):

wherein the A, B, C, D, or E ring is independently fully saturated orpartially saturated, R¹ is selected from hydrogen or a carbohydrateresidue, C2, C11, C12, and C19 are each independently substituted withhydrogen or —OH, R^(2a) and R^(2b) are selected from hydrogen, —COOH, orCOOR⁴, or together form —CO2-, R⁴ is a certain monosaccharide residue,R^(3a) and R^(3b) together form CH₂═, or are each independently selectedfrom —CH₃ or —CH₂—OH, as a stereoisomer, enantiomer or tautomer thereof,or a pharmaceutically acceptable salt thereof.

Embodiments of the present disclosure also relate to a kit for treatingdiseases, for example, a pulmonary disease. The kit may include anamount of the compound of formula (I), wherein the A, B, C, D, or E ringis independently fully saturated or partially saturated, R¹ is selectedfrom hydrogen or a carbohydrate residue, C2, C11, C12, and C19 are eachindependently substituted with hydrogen or —OH, R^(2a) and R^(2b) areselected from hydrogen, —COOH, or COOR⁴, or together form —CO2-, R⁴ is acertain monosaccharide residue, R^(3a) and R^(3b) together form CH₂═, orare each independently selected from —CH₃ or —CH₂—OH, as a stereoisomer,enantiomer or tautomer thereof, or a pharmaceutically acceptable saltthereof. In certain embodiments, the compound of formula (I) is capableof treating the pulmonary disease. In certain embodiments, the compoundof formula (I) is in a pharmaceutically acceptable carrier.

In some embodiments, the disease may include a pulmonary disease, whichmay include at least one of asthma, chronic obstructive pulmonarydisease (COPD), bronchitis, chronic or acute bronchoconstriction, adultrespiratory distress syndrome, acute lung injury, and bronchiectasis. Inparticular embodiments, the pulmonary disease may include asthma orCOPD.

Chronic obstructive pulmonary disease (COPD) includes a condition inwhich there is limited airflow in the lungs. COPD may develop andworsens over time, and result in changes in the small airways of asubject with COPD. These changes cause walls to narrow duringexpiration, making the subject hard to breathe. In certain subjects, thesmall sacs where oxygen and carbon dioxide are exchanged are destroyed,gradually starving the body of oxygen. COPD may be associated with a setof breathing-related symptoms, for example, being out of breath, chroniccough, spitting or coughing mucus (phlegm), the ability to exhale(breathe in) getting worse over time. COPD may have three forms:emphysema, chronic bronchitis, and obstructive bronchiolitis. Theemphysema may be marked by destruction of the alveoli, grape-likeclusters of air sacs at the end of the smallest airways (thebronchioles) in the lung. The chronic bronchitis may be defined ascoughing and overproduction of mucus for a threshold time during apredetermined time period. The obstructive bronchiolitis involve aninflammatory condition of the small airways. COPD may also be called aschronic obstructive airways disease, Chronic obstructive lung disease,Chronic bronchitis, Emphysema, Bronchitis-chronic. Smoking may causeCOPD while other reasons may also lead to COPD. For example, a subjectwho lacks alpha-1 antitrypsin or is exposed to certain gases, fumes ordust may develop COPD. One of the tests for COPD is a lung function testcalled spirometry, which involves blowing out as hard as possible into asmall machine that tests lung capacity. In some instances, using astethoscope to listen to lungs, pictures of lungs, blood tests may beused for determination of COPD.

Asthma includes a pulmonary condition that makes a subject with asthmadifficult to breathe properly. For example, in response to allergens orother environmental triggers, the airways of the subject may undergochanges. The changes appear to be two specific responses hyperreactiveresponse (also called hyperresponsiveness) and inflammatory response.These responses in the airways cause coughing, wheezing, shortness ofbreath (dyspnea), or other symptoms of asthma. Asthma shares some of thesymptoms of COPD (e.g., chronic coughing, wheezing, shortness of breath,etc.), and a subject may have asthma and COPD at the same time. Forexample, roughly 40% of known COPD suffers also have asthma. Asthma mayinclude various types of asthma such as, e.g., atopic asthma, non-atopicasthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchialasthma, essential asthma, true asthma, intrinsic asthma caused bypathophysiologic disturbances, extrinsic asthma caused by environmentalfactors, essential asthma of unknown or inapparent cause, non-atopicasthma, bronchitis asthma, emphysematous asthma, exercise-inducedasthma, allergen induced asthma, cold air induced asthma, occupationalasthma, infective asthma caused by bacterial, fungal, protozoal, orviral infection, non-allergic asthma, incipient asthma, wheezy infantsyndrome, bronchiolytis, etc. In particular embodiments, asthma mayinclude allergenic asthma.

Bronchitis includes bronchitis of various types, etiologies, orpathogenesis. For example, bronchitis may include acute bronchitis,acute laryngotracheal bronchitis, arachidic bronchitis, catarrhalbronchitis, croupous bronchitis, dry bronchitis, infectious asthmaticbronchitis, productive bronchitis, Staphylococcus or streptococcalbronchitis, vesicular bronchitis, etc.

Bronchiectasis includes bronchiectasis of various types, etiology, orpathogenesis. For example, bronchiectasis may include cylindricbronchiectasis, sacculated bronchiectasis, fusiform bronchiectasis,capillary bronchiectasis, cystic bronchiectasis, dry bronchiectasis,follicular bronchiectasis, etc. Bronchiectasis may include a conditionthat a subject receiving treatment of smooth muscle relaxation, whichmay also related to COPD.

Bronchconstriction may include a condition related to the constrictionof the airways in the lungs due to the tightening of the surroundingsmooth muscle which causes cough, wheezing and shortness of breath.Chronic bronchoconstriction may be caused by chronic obstructivepulmonary disease, and acute bronchoconstriction may be caused by acuteinducer of obstructive pulmonary diseases, e.g. exercise, dust, anddrug-induced bronchoconstriction.

Acute respiratory distress syndrome (ARDS), a lung syndrome that maycause inflammation of the lung parenchyma leading to impaired gasexchange with a systemic release of inflammatory mediators, causinginflammation, hypoxemia and frequently multiple organ failure.

In some embodiments, the disease may include hypertension,gastrointestinal motility, or other smooth muscle diseases.

In some embodiments, the A and B rings are independently fullysaturated, and R^(2a) and R^(2b) together form —CO2-.

In some embodiments, the carbohydrate residue is a monosaccharideresidue or an oligosaccharide residue. In some embodiments, the certaincarbohydrate residue is a monosaccharide residue or an oligosaccharideresidue. In certain embodiments, the monosaccharide may be arabinose(Ara), glucuronic acid (GlcA) or 2-deoxy-glucuronic acid, glucose (Glc),or rhamnose (Rha). In certain embodiments, the oligosaccharide residuemay be a disaccharide residue, a trisaccharide residue, or atetrasaccharide residue.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C12 and C19 are eachindependently substituted with —OH, C15 and C16 are each independentlysubstituted with two hydrogens, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atrisaccharide residue. For example, the trisaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C12 and C19 are eachindependently substituted with —OH, C15 and C16 are each independentlysubstituted with two hydrogens, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, and R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atetrasaccharide residue. For example, the tetrasaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc-(1-2)-Glc.

In certain embodiments, the A, B, C, and E rings are fully saturated,the D ring is partially saturated, the C11 and C19 are eachindependently substituted with —OH, C11 is substituted with hydrogen,R^(2a) and R^(2b) together form —CO2-, R^(3a) and R^(3b) are —CH₃, andR¹ is a monosaccharide residue or an oligosaccharide residue. Inparticular embodiments, R¹ is a trisaccharide residue. For example, thetrisaccharide residue may be -Ara-[(1-2)-Rha]-(1-3)-Glc.

In certain embodiments, the A, B, and E rings are fully saturated, the Cand D rings are partially saturated, the C19 is substituted with —OH, C9is substituted with hydrogen, R^(2a) and R^(2b) together form —CO2-,R^(3a) and R^(3b) are —CH₃, and R¹ is a monosaccharide residue or anoligosaccharide residue. In particular embodiments, R¹ is atrisaccharide residue. For example, the trisaccharide residue may be-Ara-[(1-2)-Rha]-(1-3)-Glc.

In some embodiments, the compound of formula (I) is isolated from anextract of kudingcha (KE). In certain embodiments, KE may include one ormore solutions and/or compounds extracted from kudingcha using methodsdescribed in the present disclosure and/or methods known to one ofordinary skill in the art. The kudingcha may include, for example, Ilexkudingcha, x latifolia, Ilex kaushue, Ilex pentagona, Ilex cornuta, Ilexparaguariensis, Ligustrum robustum, etc., which may be collected fromvarious places.

In some embodiments, the subject is a human. In other embodiments, thesubject may be domestic animals, such as cats, dogs, swine, cattle,sheep, goats, horses, rabbits, and the like.

In some embodiments, the method may include delivering thetherapeutically effective amount of the compound of formula (I) to anairway of a lung of the subject. In certain embodiments, the method mayinclude delivering the therapeutically effective amount of the compoundof formula (I) to an airway of a lung of the subject via inhalation ofthe compound of formula (I) by the subject.

In some embodiments, the kit may include a delivery device such as, forexample, a spray device or a pressurized delivery device.

kudingcha and Extracts of kudingcha

kudingcha may be made of various plants. For example, the plants mayinclude Ligustrum purpurascens Y. C. Yang, Ligustrum pricei Hayata,Ligustrum japonicum var. pubescens Koidz., Ligustrum delavayanumHariot., Ligustrum henryi Hemsl., Ligustrum lucidum Ait., Ilex kudingchaC. J. Tseng, Ilex latifolia Thunb, Ilex cornuta Lindl., Ilex pentagonaS. K. Chen, Ilex kaushue S. Y. Hu, Ligustrum sinense var. myrianthum(Diels) Hofk., Ligustrum robustum (Roxb.) Bl., Cratoxylum formosumsubsp. Pruniflorum (Kurz) Gogelin, Ehretia thyrsiflora (Sieb. Et. Zucc.)Nakai., Clerodendrum fortunatum Linn., Mahonia (Fort) Carrie., Iteailicifolia Oliver., etc. Among these plants, the widely used plants forkudingcha at the current time are Ilex plants, Ilex kudingcha C. J.Tseng, Ilex pentagona S. K. Chen, Ilex huoshanensis Y. H. He andLigustrum (e.g. Ligustrum robustum (Roxb.) Bl., Ligustrum henryi Hemsl.,Ligustrum pricei Hayata.

kudingcha may have various pharmaceutical activities in differentsystems. For example, in the vascular system, crude extracts ofkudingcha may improve blood circulation of heart and brain, possibly dueto the release of blood vessel tone through total saponins. In theheamoitic system, kudingcha extracts may inhibit platelet aggregation,reduce circulating lipid, regulate circulating glucose level, etc.kudingcha may also have efficacy on hyperlipidemia, and affectmetabolism of the body. When drank as tea, kudingcha may reduce bodyweight by inhibiting adopocyte production, for example, via a mechanismassociated with inhibition activity of Ilex kudingcha toward ACAT(Triterpenoidacyl CoA cholylacyl Transferase).

In the immune system, kudingcha extracts and saponins may be active inmodulating the function of macrophage, lymphocytes, thereby inhibitingdifferent inflammatory reactions. kudingcha as drinking tea may have aneffect on vessel and skin inflammation.

In the respiratory system, the extracts of Ilex latifolia Thunb mayinhibit airway smooth muscle contraction induced by external calcium,but the underlying mechanism and the identity of its active substanceremain unknown. Extracts of L. lucidum Ait are effective in eliminatingphlegm and inhibiting cough, but there is no clinical report of treatingrespiratory diseases, including asthma and other COPD diseases, with L.lucidum Ait.

In some embodiments, various groups of substances from kudingcha wereidentified, for example, a group of phenylpropanid compounds,flavonoids, terpenoids, essential oils, etc. The group of phenylpropanidcompounds include ligupurpuroside A and B, etc. The group of flavonoidsinclude quercetin, hyperoside, luteolin-7-glucoside etc. The group ofterpenoids include monterpenes and triterpenes. Also, several compoundsof triterpenes are also identified from kudingcha such as, for example,latifoloside B, C, N and O, siaresinolic acid, 24-hydroxyl-oleanolicacid, kudinoside A^(˜)T, ilekudinoside A^(˜)S, ilexoside XL VIII,oleanolic acid, beta-amyrin, olean-12-ene and kudingchagenin I. Thegroup of essential oils are produced primarily from the young leaves.

Diseases and Smooth Muscle Contractility

Asthma includes a chronic disease of the lung characterized by episodicand occasionally persistent airflow obstruction. It causes substantialmorbidity and occasional mortality among its sufferers. Nearly 300million people all over the world suffer from asthma and it has broughtstriking social losses as well as economic losses. The featuredsyndromes of asthma may include shortness of breath, chest tightness,and coughing in asthmatic episodes. These syndromes of asthma may betriggered by the exposure to allergens, cold air, exercise or otherirritants. The pathological alteration of asthma may be characterized bychronic inflammation, mucus hyperplasia, reversible airway obstruction,and airway hyperresponsiveness (AHR), in which hyperresponsiveness ofairway smooth muscle contractility is believed to be a cause of airwayconstriction. AHR may be defined as an excessive airway narrowing inresponse to a variety of chemical and physical stimulus that have littleor no effects in healthy subjects and is documented in vivo by leftwardand upward shifts of the dose-response curve to constrictor agents. Theincreased sensitivity to stimuli may be usually caused by inflammatoryreaction surrounding smooth muscles tissues. Based on the pathogenesisof asthmatic constriction, several therapeutic strategies for asthma maybe developed, including inhibition of allegoric immunological response,attenuation of airway remodeling and relaxation of airway smooth musclecontraction.

Chronic obstructive pulmonary disease (COPD) includes a condition inwhich there is limited airflow in the lungs. COPD may develop andworsens over time, and result in changes in the small airways of asubject with COPD. These changes cause walls to narrow duringexpiration, making the subject hard to breathe out. In certain subjects,the small sacs where oxygen and carbon dioxide are exchanged aredestroyed, gradually starving the body of oxygen. COPD may be associatedwith a set of breathing-related symptoms, for example, being out ofbreath, chronic cough, spitting or coughing mucus (phlegm), the abilityto exhale (breathe out) getting worse over time. COPD may have threeforms: emphysema, chronic bronchitis, and obstructive bronchiolitis. Theemphysema may be marked by destruction of the alveoli, grape-likeclusters of air sacs at the end of the smallest airways (thebronchioles) in the lung. The chronic bronchitis may be defined ascoughing and overproduction of mucus for a threshold time during apredetermined time period. The obstructive bronchiolitis involve aninflammatory condition of the small airways. COPD may also be called aschronic obstructive airways disease, Chronic obstructive lung disease,Chronic bronchitis, Emphysema, Bronchitis-chronic. Smoking may causeCOPD while other reasons may also lead to COPD. For example, a subjectwho lacks alpha-1 antitrypsin or is exposed to certain gases or fumesmay develop COPD. One of the tests for COPD is a lung function testcalled spirometry, which involves blowing out as hard as possible into asmall machine that tests lung capacity. In some instances, using astethoscope to listen to lungs, pictures of lungs, blood tests may beused for determination of COPD.

Smooth muscle contractility may be regulated by a network of signalingpathways centered on the molecular motor myosin as well as membraneproperties associated with calcium handling and cell adhesion.Depolarization of the cell membrane activates voltage-gated Ca²⁺channels, resulting in Ca²⁺ influx and activation of myosin cross-bridgecycling on actin filaments by regulatory light chain (RLC)phosphorylation catalyzed by Ca²⁺/calmodulin-dependent myosin lightchain kinase (MLCK). Agonist stimulation of G protein-coupled receptorson smooth muscle cell surfaces may recruit other regulatory elements,thereby regulating contractility through a calcium sensitizationmechanism. Smooth muscle contractility may be mediated through acalcium-dependent mechanism. The use of MLCK knockout mice shows thatmyosin light chain kinase (MLCK) is required for smooth musclecontraction, emphasizing the importance of a calcium-dependent mechanismfor smooth muscle contraction. Importantly, deletion of MLCK leads to anabolishment of asthmatic airway constriction.

The various embodiments described above may be combined to providefurther embodiments. Aspects of the embodiments can be modified, ifnecessary to employ concepts of the various patents, applications andpublications to provide yet further embodiments.

These and other changes may be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

EXAMPLES Example 1 kudingcha Extract (KE) Relaxed Evoked Force of AirwaySmooth Muscle

1000 g dried leaves of Ilex latifolia Thunb were extracted by 90-100%alcohol overnight or longer, and then remove the alcohol. The resultantpellet was dissolved in distilled water followed by ethyl acetate andn-butanol sequential extraction. The substances in ethyl acetate (EtOAcphase), n-butanol (n-BuOH phase) and water (water phase) were pelletedby dry out the solvents. The extracts from different phases weredissolved in ethanol and subjected to analyzing activity towardsrelaxation of airway smooth muscles. The leaves of kudingcha werepurchased from the Huilong pharmaceutical company, and identified byProfessor Pan Yang at Nanjing University of Chinese Medicine, P. R.China.

Briefly, the entire respiratory tree was rapidly removed and immersed inKrebs-Henseleit buffer (118.1 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 2.5 mMCaCl₂, 1.0 mM NaH₂PO₄, 25 mM NaHCO₃ and 11.1 mM glucose, pH 7.4).Bronchial rings, 2 mm in length, were isolated from pulmonary bronchiusing a dissection microscope. Then the bronchial segments were mountedin a small-vessel wire myograph chamber (Danish Myo Technology, Aarhus,Denmark) by threading on two steel wires (40 μm in diameter) secured totwo supports. One support was attached to a force transducer formeasurements of isometric force development, and the other was attachedto a micrometer controlling ring circumference. The preparation was keptin the chamber, immersed in 5 ml of Krebs-Henseleit solution, bubbledwith 5% CO₂ and 95% O₂ and maintained at 37° C. The initial isometrictension of bronchial ring was set to 0 mN prior to equilibration for 20min. The bronchial ring was then stretched in order to keep the force at5 mN followed by an additional equilibration for 20 min. The isometricforces produced by bronchial ring was recorded with a data acquisitionand analysis program (Danish Myo Technology, Aarhus, Denmark).

The robust relaxation effect and low risk of toxicity of KE on airwaysmooth muscle indicates its promising prospects for development. Toidentify the active ingredients of KE, the KE was divided into threeparts including EtOAc phase, n-BuOH phase and water phase using EtOAcand n-BuOH sequential extraction. Upon the airway smooth muscle ringsreached a relative steady contraction state evoked by MCh, accumulatedvolume of these three phases was added to assess the relaxationcapacity. As the dose increased, all these three parts can relax thebronchial rings with a dose dependent manner, but showed considerabledifferences in efficiency (FIGS. 1 A, B, C). Surprisingly, the bronchialrings started relax as little as 0.1 μL of n-BuOH phase added, and 1 μLnearly completely relaxed the contractile bronchial rings (FIGS. 1 A,D). On the other hand, 1 μL of EtOAc phase just relaxed the rings to90%. And 5 μL of extract from water phase almost had no relaxationactivity (FIG. 1D). The results suggest that n-BuOH phase is more activethan other parts of KE on relaxing airway smooth muscle. These resultsdemonstrate that KE extracts efficiently relax evoked force of airwaysmooth muscle.

Example 2 kudingcha Extract (KE) Inhibits Asthmatic Airway Constriction

Asthmatic airway constriction may be attributable to hyperresponsivesmooth muscle and tissue remodeling. To test the effect of KE on releaseof the constriction, an asthma animal model was established throughwhich evaluated the efficacy of KE extract from n-butanolphase.

Asthma mice were established as reported previously. Briefly, six toeight-week-old female C57BL/6 mice were carried out by intraperitonealinjection of 100 μg ovalbumin and 4 mg Imject Alum (ThermoFisherScientific) in total volume of 0.2 mL on days 0 and 14. These mice werethen challenged with 1.5%-2% aerosolized ovalbumin for 1 h on days 24,25 and 26. The efficacy of drugs was evaluated by measuring airwayresistance in these mice 24 h after the last challenge.

The airway resistance was measured through an invasive method asdescribed in previous reports. In brief, the mice were anesthetized with240 mg/kg Avertin and the trachea was cannulated with an 18-gauge metalneedle. Mechanical ventilation (FlexiVent; SCIREQ Inc., Montreal,Canada) was applied at a frequency of 150 breaths per minute, a tidalvolume of 10 ml/kg, and positive end-expiratory pressure of 2.5 cm H₂O.Prior to methacholine challenge, deep inflation was applied followed byadministration of aerosolized PBS to obtain stabilization of airwayresistance (Rrs, cmH₂O·s/mL). The Rrs after PBS was considered to be thebaseline. Then mice were challenged with sequential concentrations ofmethacholine with a dose of 2.0, 4.0, 8.0, 16, 32 and 64 mg/mL by anultrasonic nebulizer until reaching bronchoconstrictive phase that wasfour to five folds greater than baseline. Three minutes after the lastdose of methacholine inhalation, the reagents were delivered by the sameultrasonic nebulizer. The Rrs measurements were performed every 30 sthroughout the experiments. The negative control for these studies wasPBS, because all the solvents for reagents used here were PBS. As apositive control, albuterol (3 μg; Sigma), the most widely usedβ-agonist as an effective bronchodilator for asthma therapy, was used.

After challenge with MCh, the mice displayed sustained airwayconstriction. Upon treatment with 30 μg n-BuOH phase, the airwayresistance declined immediately within 1 minutes followed by gradualreduction. As a positive control, albuterol (3 μg) also led tosignificant reduction of airway resistance in a similar manner (FIGS. 2Aand B). These results show that n-BuOH phase efficiently releasesasthmatic airway resistance

Example 3 Identification of Active Ingredients from n-BuOH Phase

For column chromatography, silica gel (Qingdao Marine Chemical Industry,100-200 mesh) and silica gel for thin-layer chromatography (QingdaoMarine Chemical Industry, H degree) were used. Homogeneity of fractionswas determined on Thin layer Chromatography (TLC) with silica gel F,which was coated on glass plates (Qingdao Marine Chemical Industry). Thespots were detected by spraying with 10% H₂SO₄ EtOH reagent followed by105° C. heating for 5 min. Reddish-purple spots will appeared at thearea containing substances.

100 mL KE extracted with EtOAc (5×100 mL) and n-BuOH (5×100 mL)successively. The n-BuOH layer were combined and concentrated to drynessin vacuo. Then the residue was powdered (27.41 g in all) and subjected(12 g) to column chromatography (CC) on silica gel (mixture of 540 g100-200 mesh silica gel and 180 g H degree silica gel) using a stepwisegradient elution of CHCl₃-MeOH (100:0, 3.5 L), (98:2, 3 L), (95:5, 5.5L), (90:10, 33.5 L), (85:15, 20 L), (80:20, 24.5 L). Eventually, 27fractions (Fr. 1-Fr. 27) were obtained after Homogeneity.

As n-BuOH phase is still a crude extraction part, it is necessary toidentify the compound(s) with strong activity toward airway smoothmuscle relaxation. N-BuOH phase was extracted by column chromatographyand separated it into 27 fractions. For each individual fraction, thesimilar strategy was used to address the relaxation effect on bronchialrings in vitro. The former 17 fractions exhibited little effect onreducing the force even by adding 50 μL into the chamber. Strikingly,the 18th fraction (Fr. 18) showed profound effect on relaxing bronchialrings. Upon addition with 20 μL of Fr. 18 extract almost decreased theevoked force completely. Fr. 19 to Fr. 25 also had strong efficacy ofrelaxing, but was weaker than Fr. 18. The extracts from Fr. 26 and Fr.27 showed much less active to relax the rings (FIGS. 3A-C). Throughoutthe whole fractions screening, it is concluded that the putativecompound(s) might be enriched in Fr. 18 to Fr. 25. These results showthat the extract in Fr. 18 is more efficient than other ingredients withrelaxation effect, and relaxes the bronchial rings in a dose responsivemanner.

Example 4 Structural Identification and Functional Characterization forthe Active Compounds of kudingcha

To investigate identity of the active compounds, the substances infraction 18 was purified with HPLC, and then analyzed their individualstructures with NMR and others tools. In particular embodiments, theextracts were generated from Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng.

The partial purified extract was further purified on preparative HPLCusing MeOH—H₂O as a flow. Each absorption peak was collected andsubjected to spectra analysis.

NMR spectra, including ¹H NMR, ¹³C NMR, ¹H-¹H COSY, DEPT, HMBC and HSQC,were obtained in pyridine d₅ using a 500 MHz instruments. Preparativehigh performance liquid chromatography (HPLC) was carried out using asystem composed of a Waters 2545 binary gradient module, a Waters 2489UV/Visible detector, a Waters 2767 sample manager and a XBridge™ PrepC18 5 μm OBD™ Column (19×150 mm, detected at 260 nm).

After purification with HPLC, a series of compounds were obtained fromthe extracts. NMR spectra results indicated that these compounds arerelated to triterpenoid saponins including Kudinosides andIlekudinosides. Table 1 shows an example of NMR spectra data andmolecular structures of two active compounds which is similar tokudinoside A and kudinoside D. Characterization of structure of theactive compounds was summarized in FIGS. 4A-F.

Kudinosides significantly relaxed the bronchial rings in adose-dependent manner. Kudinoside D and A show highly effective amongthe triterpenoid saponins. Their dose-responsive effect on relaxation ofairway smooth muscle was measured. When the force evoked byacetylcholine reached a relatively steady state, kudinosides were addedto the bath in cumulative concentrations (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10μM, 30 μM). Both kudinoside D and kudinoside A significantly relaxedbronchial rings in a dose-dependent manner (FIGS. 5A-B). When theconcentration of kudinoside D increased to 10 μM, about 50% of the forcewas relaxed. Strikingly, upon treatment with 30 μM of kudinoside D, morethan 80% of the force was relaxed (FIG. 5B). Similarly, 30 μM ofkudinoside A also led to 80% inhibition of evoked force. Note that therelaxation effect of kudinosides was completely reversible, because therelaxed muscles restored response to MCh after washing off.

Various triterpenoid substances were identified (e.g., FIGS. 4A-F) fromIlex latifolia Thunb and Ilex kudingcha C. J. Tseng, and characterizedpreliminarily the association of structure and biological activity. Asillustrated in FIGS. 4A-F, the basic structure of pentacyclictriterpenoids is included, and side modification (e.g., the existence oflactone moiety) may affect biological activity. For example, KudinosideH fails to show effective bronchial ring relaxation.

TABLE 1 ¹³C NMR spectral data for Kudinoside A (1), C (2), D (3) and F(4) in Pyridine-d₅ (125 MHz) Aglycone Part Carbohydrate Moieties δ_(c)δ_(c) Position 1 2 3 4 Position 1 2 3 4 1 39.193 39.219 38.464 39.109 3-1 104.742 105.097 104.75 104.803 Ara 2 26.234 26.25 26.468 26.662 274.717 74.393 74.862 74.746 3 88.269 88.436 88.209 88.08 3 82.143 82.83582.145 82.197 4 39.624 39.578 39.694 39.571 4 68.198 69.421 68.19868.232 5 56.233 56.382 55.404 55.928 5 64.833 65.748 64.838 64.88 618.514 18.559 18.391 18.444 Glc 1 104.669 103.038 104.807 104.755 735.485 35.491 32.973 35.189 2 74.597 84.628 74.987 74.97 8 41.697 41.71842.222 42.825 3 78.223 78.422 78.296 78.618 9 44.902 44.895 54.57450.164 4 71.437 70.873 71.554 71.51 10 36.985 37.001 36.674 37.151 578.557 78.353 78.611 78.287 11 28.834 28.852 127.224 71.51 6 62.52862.396 62.632 62.597 12 66.06 66.075 128.429 33.521 Glc 1 106.331 13146.366 146.391 140.757 143.529 2 76.117 14 43.894 43.911 42.249 45.5453 78.19 15 28.834 28.852 25.893 29.279 4 70.504 16 26.69 26.773 26.35126.963 5 78.808 17 44.067 44.089 43.847 46.236 6 61.893 18 137.477137.459 135.051 135.742 Rha 1 101.913 100.999 101.981 101.958 19 74.30874.313 74.185 73.276 2 72.424 72.38 72.464 72.47 20 85.646 85.674 85.90885.332 3 72.523 72.507 72.582 72.563 21 28.366 28.38 28.597 29.221 473.924 73.968 73.979 73.947 22 32.826 32.831 32.973 32.505 5 70.01869.791 70.075 70.039 23 28.04 28.111 27.797 28.019 6 18.171 18.28518.489 18.601 24 16.946 17.115 16.451 17.131 25 16.731 16.771 18.61416.907 26 18.581 18.173 16.54 16.812 27 23.446 23.464 18.714 21.574 28175.37 175.403 175.179 175.339 29 25.189 25.208 23.733 26.574 30 19.45719.472 19.518 20.186

Since glycone and aglycone portions may be destroyed by hydrolysis ofacid, certain kunidnosides were treated with 2 M HCl to remove theglycone residues. 1.9 mg Kudinoside A was immersed in 20 μL 2 M HClfollowed by 95° C. boiling for 6 h, then diluted with ddH₂O andextracted with CH₃Cl. The organic and water layer were evaporated anddissolved in 20 μL DMSO. The resultant substances were subjected torelaxation assay. The aglycone portion is hydrophobic and is distributedin the organic phase. The sugars of glycone portion are hydrophilic, andglycone portion is dissolved in the water phase. As illustrated in FIGS.8A-C, HCl treatment of Kudinosides leads to significant inhibition ofrelax activity since neither the substance in organic phase nor in waterdoes phase relax the airway smooth muscle evoked by MCh stimulations.These results indicate that glycone may contribute to relaxantactivities of certain Kudinosides.

In Ilex latifolia Thunb and Ilex kudingcha C. J. Tseng, the majoraglycone of triterpenoid saponins including a C30 skeleton may contain apentacyclic structure, as illustrated in FIGS. 4A-F. These aglycone oftriterpenoid saponins may belong to ursane substitution patterns ofpentacyclic triterpenoid saponins because they contain methyl residuesboth in C19 and C20. The basic aglycone skeleton of certain Kudinosidesmay contribute to relaxation activities of these Kudinosides. Further,failure of Kudinoside H to relax airway smooth muscle strips mayindicate that the lactone moiety contributes to relaxation activities ofcertain Kudinosides. For example, pentacyclic triterpenoid saponinsbelong to a diverse group of triterpenoid saponins, and show variouspharmacological activities. As a group of pentacyclic triterpenoidsaponins, Kudinosides have relative less pharmacological activities andhave a structure of lactone moiety.

To investigate the association of lactone structure with kudinosidesactivities, 45 kinds of well-documented pentacyclic triterpenoids wereselected and subjected to relaxation assay. These pentacyclictriterpenoids include Glycyrrhetinic acid, Glycyrrhizic acid,18-B-Glycyrrhetinic acid, Saikosaponin A, Saikosaponin B1, SailosaponinB2, Saikosaponin C, Saikosaponin D, Raddeanin A, Phytolaccagenin,Esculentoside A, Bayogenin, α-Boswellic acid, Ginsenoside-Ro, AralosideX, Dipsacoside B, Akebia saponin D, Echinocystic acid, Hedera saponin B,Ciwujianoside B, Pedunculoside, Soyasaponin Ba, Soyasaponin Bb, Asiaticacid, Asiaticoside, Macranthoidin B, Momordin Ic, Demethylzeylasteral,Maslinic acid, Wilforlide A, Corosolic acid, Rotundic acid, AnemosideB4, Glycyrrhizic acid ammonium salt, Tenuifolin, Polygalacic acid,Polygalasaponin F, Madecassic acid, Madecassoside, Epifriedelanol,Sodium Aescinate, Hederagenin, α-Hederin, Hederacoside C, HederacosideD. These pentacyclic triterpenoids are from different plants and haveC30 aglycone skeleton; their modifications including hydroxylations andglycosidations are different. These substances have no intramolecularesterification modification. For example, some of substances aresaponins, and others are sapogenins. These substances may be dissolvedin DMSO with a concentration of 500 mM preparing for bronchi ringisometric assay. The results show that 5 out of 45 compounds that showedabout 50% relaxation of MCh-evoked force at a final concentration of 500μM. These five compounds contain Glycyrrhetinic acid, Saikosaponin C,Soyasaponin Bb, Raddeanin A and Phytolaccagenin. Among these fivecompounds, two compounds may relax more than 80% force. These twocompounds contain Glycyrrhetinic acid and Saikosaponin C, and are ableto relax airway smooth muscle in a dose dependent manner. However, theirIC50 values (Glycyrrhetinic acid: 84.3 μM; Saikosaponin C: >150 μM) werehigher than that of Kudinoside A and Kudinoside D. These resultsindicate that lactone moiety may contribute to relaxation activities ofcertain Kudinosides.

Lactones are cyclic esters formed by intracellular esterification of thecorresponding hydroxycarboxylic acid. Like the straight-chained esters,the lactones may be hydrolyzed by incubating in a base solution, such assodium hydroxide. The lactone moiety of Kudinosides is a δ-lactone ringwith a configuration of the 20,28β-lactone. In order to disrupt thelactone moiety of Kudinosides, 5 mg Kudinoside A were dissolved it in108 μL 0.9% NaCl solution followed by adjusting a pH to 9 with sodiumhydroxide, and then incubated the reaction mixture with shaking at 37°C. for 6 h. As the hydrolysis-condensation reaction of the lactones isreversible, the resultant substance showed significantly reducedrelaxation activity compared with native Kudinoside A. These resultsconfirm that the lactone moiety contribute to the relaxation activity ofcertain Kudinosides.

Relaxation activities from kudingcha-associated plants wereinvestigated. Table 2 illustrates indicate a range of degrees ofrelaxing activity from low (+) to high (+++++); “+/−” indicates thatplant has relatively weak relaxing activity, while “—”−” indicates thatplant has no relaxing activity.

TABLE 2 relaxation activity from Kudingcha-associated plants Plants(Latin name) Activity Ilex cornuta Lindl. & Paxton +++ Mahonia bealei(Fort.) Carr. — Ilex latifolia Thunb. +++++ Ligustrum henryi Hemsl. —Ligustrum pricei Hayata — Ligustrum japonicum Thunb. Var. — pubescensKoidz. Ligustrum lucidum W. T. Aiton — Ligustrum purpurascens Y. C. Yang+/− Ligustrum robustum (Roxb.) Blume — Ilex rotunda Thunb. +++++ Ilexzhejiangensis C. J. Tseng ++ Ilex ficoidea Hemsl. var. parvifilia S.+++++ H. Fu var. nov. ined. Ilex chinese Sims +++ Ilex macrocarpa Oliv.++ Ilex pubescens Hook. & Arn. ++++ Ilex kudingcha C. J. Tseng +++++

Example 5 The Effect of Kudinosides on Releasing Asthmatic Constrictionin Animal

Acute asthma model mice were established as reported previously.Briefly, six to eight-week-old female C57BL/6 mice were carried out byintraperitoneal injection of 100 μg ovalbumin and 4 mg Imject Alum(ThermoFisher Scientific) in total volume of 0.2 mL on days 0 and 14.These mice were then challenged with 1.5%-2% aerosolized ovalbumin for 1h on days 24, 25 and 26. The efficacy of drugs by measuring airwayresistance in these mice 24 h was evaluated after the last challenge.The airway resistance was measured through an invasive method asdescribed in previous reports. In brief, the mice were anesthetized with240 mg/kg Avertin and the trachea was cannulated with an 18-gauge metalneedle. Mechanical ventilation (FlexiVent; SCIREQ Inc., Montreal,Canada) was applied at a frequency of 150 breaths per minute, a tidalvolume of 10 ml/kg, and positive end-expiratory pressure of 2.5 cm H₂O.Prior to methacholine challenge, deep inflation was applied followed byadministration of aerosolized PBS to obtain stabilization of airwayresistance (Rrs, cmH₂O·s/mL). The Rrs after PBS was considered to be thebaseline. Then mice were challenged with sequential concentrations ofmethacholine with a dose of 2.0, 4.0, 8.0, 16, 32 and 64 mg/mL by anultrasonic nebulizer until reaching bronchoconstrictive phase that wasfour to five folds greater than baseline. Three minutes after the lastdose of methacholine inhalation, the reagents were delivered by the sameultrasonic nebulizer. The Rrs measurements were performed every 30 sthroughout the experiments. The negative control for these studies wasPBS, because all the solvents for reagents used here were PBS. As apositive control, albuterol (3 μg; Sigma), the most widely usedβ-agonist as an effective bronchodilator for asthma therapy, was used.

In PBS control, the airway resistance may reduce about 30% within 1-2minutes given PBS by inhalation in bronchoconstrictive phase, the airwayresistance reduced after inhalation, and then reached a steady state.Application of bronchodilators will cause the steady state to movedownwards, indicating a decrease in airway resistance. After inhalationof kudinoside D, the airway resistance was reduced in a dose-dependentmanner. 0.03 μg of kudinoside D caused a slight reduction while 0.3 μgor 3 μg led to a significant reduction of airway resistance as shown inFIG. 6A. At 1 minute after inhalation, 0.03 μg, 0.3 μg and 3 μg ofkudinoside D reduced airway resistance to 69.65±5.87%, 67.79±6.77% and45.81±6.87% respectively, whereas 3 μg of albuterol led to a reductionof 55.59±13.07%. Surprisingly, at 3 minutes after inhalation, both 0.3μg and 3 μg kudinoside D showed more reduction of airway resistance thanthat of 3 μg of albuterol. The dose-responsive effect of kudinoside D isshown in FIG. 6B. These results indicate that kudinoside D is a strongbronchodilator, even much stronger than albuterol.

To test immunological alteration of the asthmatic animals receivedtreatment, histology analysis of the lung were conducted. Both groups ofmice received either vehicle or kudinoside D showed comparablehistological alterations including hemorrhage, inflammatoryinfiltrations of neutrophils and esiophilic cells etc. These resultsshow that inhibition effect by kudinoside D was not contributed tofailure of asthma model establishment.

The therapeutic efficacy of kudinoside C was measured and showed strongrelaxation effect on airway smooth muscles. It also showed significantinhibition of airway constriction and its IC50 value was 150 μg.Kudinoside H, exhibited little effect on relaxation in vitro, had littleinhibition activity of airway constriction in vivo, either. Theseresults indicate that other kudinosides with relaxation activity invitro may have inhibition effect on airway constriction in vivo and viceversa.

Example 6 Kudinosides Relax Airway Smooth Muscle Through Modulation ofCalcium Influx

Calcium image (1-3): Ca²⁺ signal was measured by recording 488 nm laserexcited Fluo-4 fluorescence with a laser confocal microscope (FV-1000,Olympus). After 3 times washing with D-Hanks solution, primary culturedairway smooth muscle cells were loaded with 2.5 μM Fluo-4 AM and 0.02%pluronic acid (F127) in D-Hanks for 30 min at room temperature. Thenwashed 3 times with D-Hanks, incubated in H-T buffer at 37° C. for 40min. Ca²⁺ signal was measured by recording 488 nm laser excited Fluo-4fluorescence with a laser confocal microscope (FV-1000, Olympus).

To assess the requirement of calcium reduction for kudinoside-mediatedrelaxation, smooth muscle tissues permeable were made. Briefly, thefresh bronchial ring were separated by sharp dissection from C57BL/6Jmice. Following incubation for 10 minutes in H-T buffer, the musclestrips were treated for 5 minutes in Ca²⁺-free H-T buffer and for 5minutes in buffer A (30 mmol/L TES, 0.5 mmol/L dithiothreitol, 50 mmol/LKCl, 5 mmol/L K₂EGTA, 150 mmol/L sucrose, pH 7.4), and then weresubsequently permeabilized in α-toxin (16,000 units/mL; Sigma) in bufferA for 40 minutes at room temperature.

Treatment with 10 mmol/L ionomycin (Sigma) in buffer A for 10 minuteswas used to deplete Ca²⁺ stores. Permeable tissues were washed in pCa9.0 solution (20 mmol/L TES, 4 mmol/L K₂EGTA, 5.83 mmol/L MgCl₂, 7.56mmol/L potassium propionate, 3.9 mmol/L Na₂ATP, 0.5 mmol/Ldithioerythritol, 16.2 mmol/L phosphocreatine, 15 U/mL creatine kinase,pH 6.9), followed by incubation in pCa 4.5 solution (20 mmol/L TES, 4mmol/L CaEGTA, 5.66 mmol/L MgCl₂, 7.53 mmol/L potassium propionate, 3.9mmol/L Na₂ATP, 0.5 mmol/L dithioerythritol, 16.2 mmol/L phosphocreatine,15 U/mL creatine kinase, pH 6.9) to elicit a sustained Ca²⁺-inducedcontraction. After wash out the solution, permeable muscle strips werethen incubated with pCa 5.0 solution and relaxed by adding Kudinoside Ddissolved in pCa 5.0 solution.

At rest condition, the smooth muscle cells showed weak but constantcalcium signals. Upon addition of acetylcholine, the calcium signalbecame strong with a robust followed by declining to basal level, andsmooth muscle contraction could be also detected during this process(FIG. 7). After pretreatment with kudinoside, however, the smooth muscleshowed neither elevation of calcium signal nor cellular contraction(FIG. 7). These results indicate that kudinosides may reduce calciuminflux during smooth muscle relaxation.

If the reduction of cytosolic calcium underlies the relaxation effect ofkudinoside, fixation for cytosolic calcium concentration expectedlyabolished the relaxation. When the muscle strip was skinned, pCa 4.5elicited a sustained contraction. Treatment with kudinoside D could notrelax this sustained contraction, and contrarily slightly enhanced it.These results shows that Kudinoside D did not relax smooth muscle atconstant concentration of calcium. These results also indicate thatmodulation of calcium influx is required for the relaxation bykudinosides, and the mechanism underlying relaxation of kudinosides isinvolved modulation of calcium influx.

The augmented Ca²⁺ in smooth muscle cells may be a second messenger forcontraction initiating by both electromechanical coupling andpharmocomechanical coupling. And in these two general kinds ofexcitation, L-type calcium channel plays a role in generating the influxof Ca²⁺ during contraction. So to identify the target of certainkudinosides, patch-clamp studies were used to examine the modulation ofL-type calcium channel currents after Kudinosides treatment. Inpatch-clamp studies, whole-cell patch clamp recording of VDCC currentswere performed at room temperature, in response to voltage pulses (−60to +50 mV from a holding potential of −80 mV, 200 ms). 50 μM ofKudinoside A can decrease about 60% of the current generated by L-typecalcium channel compared to the vehicle (FIGS. 9A-B). This resultsindicate that L-type calcium channel may be an essential target ofcertain kudinosides. However, as a positive control, 1 μM of Nifedipine,a well-established antagonist of L-type calcium channel, can almosteliminate the current of L-type calcium channel (FIGS. 9A-B). There maybe more other targets of Kudinosides remaining to be determined.

Example 7 Outcome of kudingcha Extract in Human Asthma

Volunteer 20 to 50 years of age were eligible for enrollment if they hadmoderate asthma, diagnosed according to the guidelines of the GlobalInitiative for Asthma (See Table 3). All the patients require therapywith inhaled corticosteroids and beclomethasone or long-actingbeta₂-adrenergic agonists or salmeterol or the equivalent to maintainreasonable control during asthma episode.

10 subjects who had been treated with inhaled therapy were randomlyassigned. Because the volunteers with asthma symptoms are moderate andreversible or partial reversible, the individual person before treatmentwas designed as a control. The primary outcome was the acute release ofdyspnea.

TABLE 3 Demographic and Clinical characteristics of subjects completing2 weeks treatment Characteristic Note No. of subjects 10 8 male/2 femaleAge-yr 26-51 Asthma severity moderate 6/mild 4 Seasonal allergies 10present

TABLE 4 Acute outcome of subjects receiving once treatment aftertreatment Event before treatment 5 minute 30 minutes Dyspnea 10 0 0Wheezing 3 0 0 Cough 8 3 Chest discomfort 5 3 3 Nasal congestion 3 3 3

Ten volunteers are from West-North and South of China where show asignificant geological difference in seasons, culture and living styles.All the patients received 1-2 sprays of KE (55 mg/mL) treatment duringasthma episode. Dyspnea and wheezing symptoms were significantlyattenuated within 5 minutes, and no recurrence was observed within 30minutes (Table 4). In most cases (8/10), the volunteers did not needsecond round of spray treatment within 12 hours. KE treatment alsoattenuated cough frequency after once spraying, but no inhibition ofscreatant production in airway and nose was observed (p>0.05). Aninteresting outcome is that the bronchitis may be inhibitedsignificantly in 4/10 volunteers after repeated treatment with KE.During the treatment periods, there is no any side-effect in terms ofbreath, vomiting, allergy, dry mouth as well as other feeling. Theseresults show that KE extract displays significant efficacy on releasedyspnea of asthma, showing an acute complete response.

In order to test the effect of single substance in KE extract on asthmatherapy, two volunteers (male 51 y; female 72 y) with severe asthmasyndromes were treated with kudinoside A (20 mM) with an atomizationdevice. After inhalation for 15 seconds, the male showed release ofdyspnea at 5 minutes, and PEF value increased from 100 to 320. Therelease was maintained overnight. As a control of albuterol, this malevolunteer showed release of dyspnea within 3 minutes, but the releasecould not be maintained overnight, and once more inhalation wasrequired. For the female volunteer, similar result was obtained. Thisresult shows that as one of active components of KE, kudinoside A alsodisplays significant efficacy on release dyspnea of asthma.

What is claimed is:
 1. A method for treating a pulmonary disease comprising administering to a subject in need thereof a therapeutically effective amount of an active ingredient consisting of a compound of formula (I) or stereoisomer, enantiomer, tautomer or a pharmaceutically acceptable salt thereof:

wherein: the A, B, C, D, or E ring is independently fully saturated or partially saturated; R¹ is a carbohydrate residue; the positions of C2, C11, C12, and C19 are each independently substituted with hydrogen or —OH; R^(2a) and R^(2b) together form —CO₂—; R^(3a) and R^(3b) together form CH₂, or are each independently selected from —CH₃ or —CH₂—OH.
 2. The method of claim 1, wherein the pulmonary disease includes at least one of asthma, chronic obstructive pulmonary disease, bronchitis, chronic or acute bronchoconstriction, adult respiratory distress syndrome, acute lung injury, and bronchiectasis.
 3. The method of claim 1, wherein the carbohydrate residue is a monosaccharide residue or an oligosaccharide residue.
 4. The method of claim 3, wherein: the monosaccharide residue is selected from the group consisting of arabinose, glucuronic acid, 2-deoxy-glucuronic acid, glucose, and rhamnose; and/or the oligosaccharide residue is a disaccharide residue, a trisaccharide residue, or a tetrasaccharide residue comprising any combination of glucose, arabinose, rhamnose and glucuronic acid.
 5. The method of claim 1, wherein: the A, B, C, and E rings are fully saturated; the D ring is partially saturated; the positions of C12 and C19 are each independently substituted with —OH; R^(3a) and R^(3b) are each independently —CH₃; and R¹ is a monosaccharide residue or an oligosaccharide residue; or the A, B, C, and E rings are fully saturated; the D ring is partially saturated; the positions of C11 and C19 are each independently substituted with —OH; R^(3a) and R^(3b) are each independently —CH₃; and R¹ is a monosaccharide residue or an oligosaccharide residue; or the A, B, and E rings are fully saturated; the C and D rings are partially saturated; the position of C19 is substituted with —OH; R^(3a) and R^(3b) are each independently —CH₃; and R¹ is a monosaccharide residue or an oligosaccharide residue; or the compound of formula (I) is selected from:

wherein R¹ is a monosaccharide residue or an oligosaccharide residue.
 6. The method of claim 5, wherein: the monosaccharide residue is selected from the group consisting of arabinose, glucuronic acid, 2-deoxy-glucuronic acid, glucose, and rhamnose; and/or the oligosaccharide residue is a disaccharide residue, a trisaccharide residue, or a tetrasaccharide residue comprising any combination of glucose, arabinose, rhamnose and glucuronic acid.
 7. The method of claim 1, wherein the compound of formula (I) is selected from the group consisting of kudinoside A, kudinoside B, kudinoside C, kudinoside D, kudinoside E, kudinoside F, kudinoside I, kudinoside J, Ilekudinoside H, Ilekudinoside I, and Ilekudinoside J.
 8. The method of claim 1, wherein the compound of formula (I) is isolated from an extract of kudingcha.
 9. The method of claim 1, wherein the subject is a human being or domestic animal.
 10. The method of claim 1, wherein the administering comprises delivering the compound of formula (I) or stereoisomer, enantiomer, tautomer or a pharmaceutically acceptable salt thereof to the airway of the subject via inhalation.
 11. The method of claim 1, wherein the compound of formula (I) or stereoisomer, enantiomer, tautomer or a pharmaceutically acceptable salt thereof is in a pharmaceutically acceptable carrier or in a delivery device.
 12. The method of claim 1, wherein the compound of formula (I) or stereoisomer, enantiomer, tautomer or a pharmaceutically acceptable salt thereof forms an inhalation dosage form.
 13. The method of claim 1, wherein the compound of formula (I) or stereoisomer, enantiomer, tautomer or a pharmaceutically acceptable salt thereof forms an aerosol form.
 14. The method of claim 11, wherein the delivery device is a spray device or a pressurized delivery device. 